The cell-free protein synthesis system derived from cultured mammalian cells is important for analysis of gene products in eukaryotes and research of translational regulation. Most generally, rabbit reticulocyte lysate is used, and a number of proteins expressed in the system exhibit normal activities in vivo, through a correct folding and processing of those proteins (for example, see Non-Patent Document 1). Post-translational modification such as acetylation, isoprenylation or phosphorylation is required in some cases for the function or activity of proteins. Alternative post-translational modification (processing) such as cleavage of a signal peptide or addition of a carbohydrate chain has been achieved by adding a canine microsomal membrane to the above-mentioned translation reaction mixture (for example, see Non-Patent Document 2).
On the other hand, it is known that in a synthetic reaction of proteins in eukaryotes, particularly translation reaction, a far larger number of translation initiation factors are involved than in prokaryotes and their interaction is complicated. In initiation of the protein synthetic system in eukaryotes, at least ten members of eukaryotic initiation factors (eIFs) are required. In the first step in which a pre-initiation complex is formed, the GTP type of eIF2 transports an initiation tRNA (Met-tRNAi) to a small subunit of 40S ribosome. Then, the pre-initiation complex attaches to the 5′-end of eukaryotic mRNA. In this step, eIF4F (also termed as cap-binding complex consisting of eIF4E, eIF4A and eIF4G) and eIF3 are required. This complex searches for AUG nearest at the 5′-end. After binding of Met-tRNAi with the initiation AUG, the initiation factor eIF5 releases eIF2 and eIF3. Finally, a 60S subunit binds to the complex comprising the initiation tRNA, mRNA and 40S subunit to forma 80S initiation complex. It is considered that in eukaryotic cells the translation reaction is mainly regulated by such initiation factors (for example, see Non-Patent Documents 3-5).
In eukaryotic cells, it is known that mRNA as a template for protein synthesis has a cap structure (m7GpppN) in which 7-methylguanosine binds to the 5′-end phosphate via pyrophosphate. This structure plays an important role not only in a protective action against enzymatic degradation of mRNA but also in a mechanism of initiation of translation. The translation is promoted by binding of a cap binding factor (eIF4E) to the cap structure. At the 3′-end of mRNA, there is a poly-A structure linked by 80-100 polyadenylic acids to stabilize mRNA, which structure interacts with a translation-initiating complex through a poly A binding factor (PABP). It is known that this cap structure promotes translation in a cell-free system for protein synthesis, too. In order to provide a cap structure at the 5′-end of mRNA in vitro, a cap-like material designated as cap analogue is used as a substrate for synthesizing a cap-carrying mRNA by enzymatic reaction with RNA polymerase. When this capped mRNA is used to synthesize a protein, however, since the coexisting cap analogue has an inhibitory action to the protein synthesis, an additional manipulation for removing the cap analogue from mRNA preparation is required. In addition, there is a problem in using a cap analogue that the amount of synthesized mRNA is reduced.
Non-Patent Document 1: Pelham, H R. and Jackson, R J., “An efficient mRNA-dependent translation system from reticulocyte lysates”, (1976) Eur J Biochem. Vol. 67, pp. 247-256.
Non-Patent Document 2: Walter, P. and Blobel, G. (1983) Method, Enzymol., Vol. 96, pp. 84
Non-Patent Document 3: Imataka, H. et al., (1997) EMBO J. Vol. 16, pp. 817-825
Non-Patent Document 4: Imataka, H. et al., (1998) EMBO J. Vol. 17, pp. 7480-7489
Non-Patent Document 5: Svitkin, Y V, et al., (2001) RNA Vol. 7, pp. 1743-1752